Soybean is an important crop and is a primary food source in many areas of the world. The methods of biotechnology have been applied to soybean for improvement of agronomic traits and the quality of the product. Examples of agronomic traits introduced into soybean plants include herbicide resistance and insect resistance.
The aad-12 gene (originally from Delftia acidovorans) encodes the aryloxyalkanoate dioxygenase (AAD-12) protein. This gene confers tolerance to 2,4-dichlorophenoxyacetic acid, for example, and to pyridyloxyacetate herbicides. The aad-12 gene, itself, for introducing herbicide tolerance in plants was disclosed in WO 2007/053482.
The expression of heterologous or foreign genes (transgenes) in plants is influenced by where the foreign gene is inserted in the chromosome. This could, for example, be due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site (Weising et al., Ann. Rev. Genet 22:421-477, 1988). Thus, the same gene in the same type of transgenic plant (or other organism) can exhibit a wide variation in expression level amongst different events. There may also be differences in spatial or temporal patterns of expression. For example, differences in the relative expression of a transgene in various plant tissues may not correspond to the patterns expected from transcriptional regulatory elements present in the gene construct introduced into the plant.
Thus, large numbers of events are often created and screened in order to identify an event that expresses an introduced gene of interest to a satisfactory level for a given purpose. For commercial purposes, it is common to produce hundreds to thousands of different events and to screen those events for a single event that has desired transgene expression levels and patterns. An event that has desired levels and/or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions.
It would be advantageous to be able to detect the presence of a transgene and/or genomic DNA of a particular plant in order to determine whether progeny of a sexual cross contain the transgene and/or genomic DNA of interest. In addition, a method for detecting the presence of the transgene and/or genomic DNA in a particular plant would be helpful when complying with regulations requiring the pre-market approval and labeling of foods derived from the recombinant crop plants.
It is possible to detect the presence of a transgene by any well known nucleic acid detection method. Examples include the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, or other commonly used genetic elements. Such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA adjacent to the inserted DNA (“flanking DNA”) is known.
It is also important to be able to be able to determine the zygosity of a transgenic event in plants for breeding, trait introgression, and seed purity purposes. For regulatory and seed quality reasons, it is also advantageous to be able to rapidly detect the presence of mixed aad-12 gene events (intended- or unintended) in soybean plant material.